Enzymatic digestion of microalgal biomass for lipid, sugar, and protein recovery

ABSTRACT

Methods for the recovery of lipids, sugars, and proteins from microbial biomass by enzymatic digestion are disclosed. The methods involve treating microalgae with a fungal acid protease, or with a mixture of at least one protease and at least one amylase.

RELATED APPLICATIONS

This application claims the benefit of the PCT/US2014/053421 filed Aug.29, 2014, which claims priority to U.S. Provisional Application Ser. No.61/871,997 filed under 35 U.S.C. § 111(b) on Aug. 30, 2013, and U.S.Provisional Application Ser. No. 61/877,497 filed under 35 U.S.C. §111(b) on Sep. 13, 2013, the entire disclosures of which areincorporated herein by reference for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant numberCHE1230609 awarded by National Science Foundation and Grant numberDE-EE0005993 awarded by the United States Department of Energy. Thegovernment has certain rights in this invention.

BACKGROUND OF THE INVENTION

Rising petrochemical prices and interest in reducing CO₂ emissions haveprompted the development of bio-based chemical production. As oneexample, succinate is an important platform chemical for the productionof various high value-added derivatives such as 1,4-butanediol, ethylenediamine disuccinate, and adipic acid. Currently, succinate can beproduced from either petrochemical synthesis or microbial fermentation,the latter process having a cost that could compete favorably withpetrochemical production in the future. For bio-based succinic acidproduction, looking for the inexpensive feedstocks and optimization ofthe pretreatment process are important challenges in reducing the costof the succinic acid production. Biological pretreatment methods, suchas enzyme hydrolysis, have replaced the traditional acid hydrolysis dueto the mild conditions, fewer by-products, and lack of corrosion issues.However, there remain many challenges and an unmet need in the art formore effective and cost-efficient methods of enzyme hydrolysis.

SUMMARY OF THE INVENTION

Provided herein is a method of enzymatic hydrolysis that comprises thesteps of treating microalgae with enzymes to produce digested biomass,wherein the enzymes comprise a mixture of at least one protease and atleast one amylase, and separating the digested biomass into an organicphase and an aqueous phase, wherein the organic phase contains lipidsand the aqueous phase contains solids, carbohydrates, and peptides.

In certain embodiments, the method includes further processing and/orseparating one or both of the organic phase and the aqueous phase toobtain lipids, solids, or bio-based products. In particular embodiments,the further processing and/or separating comprises subjecting theorganic phase to a lipid-solvent separation to recover lipids. Inparticular embodiments, the further processing and/or separatingcomprises subjecting the aqueous phase to a solid-liquid separation toobtain separated solids and a supernatant, the supernatant containingcarbohydrates and nutrients, and subjecting the supernatant to microbialfermentation to obtain a bio-based product. In certain embodiments, thebio-based product comprises succinic acid. In particular embodiments,the further processing and/or separating comprises subjecting theaqueous phase to microbial fermentation to obtain a bio-based product.In certain embodiments, the bio-based product comprises succinic acid.

In certain embodiments, the method further comprises extracting lipidsfrom the solids with an organic solvent. In certain embodiments, themicroalgae is lipid-rich wet microalgae.

Provided herein is a method of enzymatic hydrolysis comprising the stepsof treating microalgae with one or more proteases to obtain digestedbiomass; separating the digested biomass into an organic phase and anaqueous phase, wherein the organic phase contains lipids and the aqueousphase contains solids, carbohydrates, and peptides; and treating atleast a portion of the aqueous phase with one or more amylases to obtainan amylase-treated aqueous phase containing hydrolyzed carbohydrates andproteins.

In certain embodiments, the method comprises further separating and/orprocessing any of the organic phase, aqueous phase, or amylase-treatedaqueous phase to obtain lipids, solids, or bio-based products. Inparticular embodiments, the further separating and/or processingcomprises subjecting either the aqueous phase or the amylase-treatedaqueous phase to microbial fermentation to obtain bio-based products. Inparticular embodiments, the further separating and/or processingcomprises subjecting the aqueous phase or the amylase-treated aqueousphase to a solid-liquid separation to obtain solids and supernatant, andsubjecting the supernatant to microbial fermentation to obtain bio-basedproducts.

Further provided is a method of enzymatic hydrolysis comprising treatingmicroalgae with an enzyme to product digested biomass, wherein theenzyme comprises a fungal acid protease, and separating the digestedbiomass into an organic phase and an aqueous phase, wherein the organicphase contains lipids and the aqueous phase contains solids,carbohydrates, and peptides. In certain embodiments, the methodcomprises further processing and/or separating one or both of theorganic phase and the aqueous phase to obtain lipids, solids, orbio-based products. In particular embodiments, the further processingand/or separating comprises subjecting the organic phase to alipid-solvent separation to recover lipids. In particular embodiments,the further processing and/or separating comprises subjecting theaqueous phase to a solid-liquid separation to obtain separated solidsand a supernatant, the supernatant containing carbohydrates andnutrients; and subjecting the supernatant to microbial fermentation toobtain a bio-based product.

Further provided is a method of enzymatic hydrolysis comprising thesteps of treating lipid-lean microalgae with a mixture of at least oneprotease and at least one amylase to obtain digested biomass; andfurther separating and/or processing the digested biomass to obtain abio-based product. In certain embodiments, the further separating and/orprocessing comprises separating solids from liquid to obtain solids anda supernatant, the supernatant containing carbohydrates and nutrients;and subjecting the supernatant to microbial fermentation to obtain abio-based product. In certain embodiments, the further processingcomprises subjecting the digested biomass to microbial fermentation toobtain a bio-based product.

Further provided is a method of enzymatic hydrolysis involving a singlestep in which the release of lipids and breakdown of polysaccharidesinto simple fermentable sugars occurs simultaneously. The methodutilizes an enzyme mixture comprising proteases, amylases, or acombination thereof. In certain embodiments, the method requires nopretreatment (thermal or mechanical) prior to enzymatic hydrolysis, andcan be conducted at low temperatures using relatively simple equipment.

Further provided is a two-stage enzymatic hydrolysis method to extractlipids and hydrolyze sugars. In the first stage, a first set of enzymesis used to disrupt the cell wall, extract lipids, and at least partiallyhydrolyze the carbohydrates. In the second stage, a second set ofenzymes is used to complete the hydrolysis of carbohydrates. In someembodiments, over 85% of lipids are extracted and 99% of the monomersugars are released.

Further provided is a one-stage enzymatic hydrolysis method to extractlipids and hydrolyze carbohydrates simultaneously. The method involvesusing a mixture of enzymes comprising proteases, amylases, or acombination thereof.

Further provided is a method of fermentation, the method comprisingusing a mixture of enzymes to hydrolyze proteins in microalgae biomassand release nutrients into the hydrolysate, wherein the releasednutrients and amino acids are used as a nitrogen source in fermentationwithout any further addition. In certain embodiments, solid residue iscollected and optional steps can be performed to extract valuableproducts therein. In certain embodiments, the liquid phase of themicroalgae hydrolysate is used in succinic acid fermentation without anyfurther addition.

Further provided is a method of conducting succinic acid fermentation,the method comprising simultaneously disrupting the cell wall, releasingcarbohydrates, and hydrolyzing the released carbohydrates into monomersugars, all while using a mixture of enzymes from lipid-free microalgae.In certain embodiments, the method does not require any pretreatmentprior to enzymatic hydrolysis and can be conducted at low temperaturesusing relatively simple equipment. This significantly reduces the costof operation. In some embodiments, over 99% of the monomer sugar can bereleased under optimized conditions.

In certain embodiments, hydrothermal treatment is used to increase theaccessibility of the enzymes to the binding sites on thepolysaccharides, which significantly improves the enzyme activity andreduces the enzyme loading.

In certain embodiments, the mixture of enzymes hydrolyzes some of theprotein content in the microalgae biomass and also releases othernutrients into the hydrolysate. The hydrolysate with released nutrientsand amino acids (a nitrogen source) is used during the fermentationprocess of bio-based products without any further addition. The solidresidue is collected and further steps can optionally be performed toextract valuable product in the solid residue. While using the liquidphase of microalgae hydrolysate in the succinic acid fermentation ofcertain embodiments, a similar yield (˜72%, w/w) and activity isachieved between the fermentation with or without supplemental yeastextract.

Further provided is a method of enzyme hydrolysis comprising treatinglipid-rich wet microalgae with proteases to produce digested biomassseparable into an organic phase and an aqueous phase, wherein theorganic phase comprises lipids and the aqueous phase comprisesundigested solids and solubilized carbohydrates and peptides; andtreating the aqueous phase with amylases to produce hydrolyzedcarbohydrates and proteins. In certain embodiments, the method furthercomprises a separation step to separate the lipids from the organicphase. In certain embodiments, the aqueous phase is microbiallyfermented into bio-based products.

In certain embodiments, the method further comprises separating theundigested solids from the solubilized carbohydrates and peptides. Incertain embodiments, the separated carbohydrates and peptides aremicrobially fermented into bio-based products.

In certain embodiments, the undigested solids are capable for use asanimal feed or fertilizer. In certain embodiments, the hydrolyzedcarbohydrates and proteins are fermented to produce bio-based products.In certain embodiments, the method further comprises separating solidsfrom the hydrolyzed carbohydrates and proteins, wherein the separatedsolids are usable for animal feed or fertilizer.

Further provided is a method of enzymatic hydrolysis comprising treatinglipid-rich wet microalgae with an enzyme cocktail comprising proteasesand amylases to produce digested biomass; and separating the digestedbiomass into an organic phase and an aqueous phase, wherein the organicphase comprises lipids and the aqueous phase comprises undigested solidsand solubilized carbohydrates and peptides. In certain embodiments, themethod further comprises separating the organic phase into lipids andsolvent, wherein the lipids are usable to produce fuels and otherproducts.

In certain embodiments, the method further comprises microbiallyfermenting the aqueous phase to produce bio-based products. In certainembodiments, the method further comprises separating the aqueous phaseinto solids and a supernatant containing carbohydrates and nutrients,wherein the solids are usable for animal feed or fertilizer. In certainembodiments, the method further comprises microbially fermenting thesupernatant to produce bio-based products.

Further provided is a method of enzymatic hydrolysis comprising treatinglipid-lean wet algae with one of proteases or a combination of proteasesand amylases, to produce digested biomass. In certain embodiments, thelipid-lean wet algae is thermally treated before being treated withenzymes. In certain embodiments, the method further comprisesmicrobially fermenting the digested biomass to produce bio-basedproducts. In certain embodiments, the method further comprisesconducting a separation step on the digested biomass to produce solidsand a supernatant, wherein the solids are usable for animal feed orfertilizer, and the supernatant comprises carbohydrates and nutrients.In certain embodiments, the method further comprises microbiallyfermenting the supernatant to produce bio-based products.

Further provided are the products of any of the methods describedherein. In certain embodiments, the products comprise succinic acid.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains one or more drawings executed incolor and/or one or more photographs. Copies of this patent or patentapplication publication with color drawing(s) and/or photograph(s) willbe provided by the U.S. Patent and Trademark Office upon request andpayment of the necessary fees.

FIG. 1: Non-limiting example of a two-stage enzymatic hydrolysis oflipid-rich microalgae using proteases and amylases, in accordance withthe present disclosure. The block flow diagram shows the process forproduction of sugar-rich hydrolysate and lipids from lipid-containingmicroalgae biomass with two stages of enzymatic hydrolysis. Recoveredlipids may be used for production of fuels and other oleochemicals whilethe released sugars may be fermented (with or without solids separation)to bio-products such as alcohols, organic acids, or methane.

FIG. 2: Non-limiting example of a one-stage enzymatic hydrolysis oflipid-rich microalgae using a mixture of proteases and amylases. Theblock flow diagram shows the process for the production of sugar-richhydrolysate and lipids from lipid-containing microalgae biomass with onestage enzymatic hydrolysis. Recovered lipids may be used for productionof fuels and other oleochemicals while the released sugars may befermented (with or without solids separation) to bio-products such asalcohols, organic acids, or methane.

FIG. 3: Non-limiting example of enzymatic hydrolysis of lipid-richmicroalgae using either a mixture of protease and amylases or proteasealone. Following enzyme treatment, the hydrolyzate is first separatedinto residual solids and supernatant. The residual solids undergosolvent extraction to recover lipids while the supernatant containinghydrolyzed sugars and protein is fermented into bio-products such asalcohols, organic acids, or methane.

FIG. 4: Non-limiting example of enzymatic hydrolysis of lipid-leanmicroalgae using protease alone or in combination with amylases.Following enzyme treatment, the hydrolysate may be fermented (with orwithout prior separation of insoluble solids) to bio-products such asalcohols, organic acids, or methane.

FIGS. 5A-5B: A fraction of lipid extractable after enzymatic treatmentof SLA-04 (FIG. 5A) and SR-21 (FIG. 5B) after enzymatic digestion withprotease (open bars) or a mixture of protease and amylases (filledbars). The horizontal dashed line indicates the total fatty acid methylester (FAME) content of the biomass samples—35% (w/w) for SLA-04 and 44%(w/w) for SR21.

FIG. 6: Photograph showing lipid droplets formed on the surface of thewater phase in enzymatically digested SLA-04.

FIGS. 7A-7B: A non-limiting example GC chromatogram of solvent extractsobtained after digestion and recovery of lipids from SLA-04 (FIG. 7A)and SR-21 (FIG. 7B). This shows that most of the recovered lipids weretriglycerides (retention time>30 min).

FIG. 8: Graph showing that a portion of lipids can remain stuck toresidual solids following enzymatic digestion, and can be extractedseparately. The y-axis shows the mass of lipids recovered from residualsolids relative to the initial mass (feed) of algae biomass.

FIGS. 9A-9B: Concentrations of solubilized sugars released from SLA-04(FIG. 9A) and SR-21 (FIG. 9B) after enzymatic digestion with protease(open bars) or a mixture of protease and amylases (filled bars).

FIG. 10: Image of a PAGE electrophoresis gel for commercial protease,α-amylase, and glucoamylase enzyme preparations. The image shows thatsome α-amylase activity might be present in the protease preparation(see overlapping band at ˜50 kD in lanes 1 and 2).

FIG. 11: Effect of the enzyme cocktail composition on sugar release.Experiments were performed using a 16% (w/v) microalgae biomass solutionfor 2 h with different combinations of α-amylase, glucoamylase, andprotease. The y-axis shows release of monomeric sugars relative to thetotal carbohydrate initially present in the biomass.

FIG. 12: Sugar release during digestion of algal biomass with varyingloadings of α-amylase (0.5-15 kU) and fixed glucoamylase loadings (150U). Experiments were performed using a 16% (w/v) microalgae biomasssolution at a pH of 4.5 and 55° C. The y-axis shows release of monomericsugars relative to the total carbohydrate initially present in thebiomass.

FIG. 13: Sugar release as a function of pH during digestion of algalbiomass with α-amylase (1 kU) and glucoamylase loadings (150 U).Experiments were performed using a 16% (w/v) microalgae biomass solutionat 55° C. The y-axis shows release of monomeric sugars relative to thetotal carbohydrate initially present in the biomass.

FIG. 14: Sugar release during digestion of algal biomass with varyingloadings of protease (0-3 kU) and fixed glucoamylase loadings (150 U).Experiments were performed using a 16% (w/v) microalgae biomass solutionat a pH of 4.5 and 55° C. The y-axis shows release of monomeric sugarsrelative to the total carbohydrate initially present in the biomass.

FIG. 15: Sugar release during digestion of algal biomass with varyingloadings of protease (0-3 kU) and fixed glucoamylase loadings (150 U).Experiments were performed using a 16% (w/v) microalgae biomass solutionat a pH of 4.5 and 55° C. The y-axis shows release of monomeric sugarsrelative to the total carbohydrate initially present in the biomass.

FIGS. 16A-16B: Fermentation of digested microalgal biomass (FIG. 16A)and glucose to succinic acid (FIG. 16B).

DETAILED DESCRIPTION OF THE INVENTION

Throughout this disclosure, various publications, patents, and publishedpatent specifications are referenced by an identifying citation. Thedisclosures of these publications, patents and published patentspecifications are hereby incorporated by reference into the presentdisclosure in their entirety to more fully describe the state of the artto which this invention pertains.

Microalgae can serve as feedstocks for economically viable andenvironmentally sustainable biofuels due to their high productivity evenwhile using low quality land, water, and nutrients. Unlike terrestrialplants, microalgae have a unicellular structure without lignin, whichenables the breakdown of the cell wall and the release of carbohydrateswith much milder pretreatment methods. As a result, microalgae do notgenerate significant amounts of inhibitors that cause degradation ofcarbohydrates during the process. Previously, microalgae have beensubjected to mechanical disruption, thermolysis, microwave, andsonication methods to disrupt the cell wall. However, all of thesemethods require great energy consumption or higher equipment cost. Thepresent disclosure describes the development of a cost-effective processwith a low-energy requirement and easy setup of equipment that, incertain embodiments, simultaneously (1) disrupts the cell walls, (2)extracts the lipids, (3) releases and hydrolyses the polysaccharides tomonomer sugars, and (4) releases and hydrolyses the proteins for anitrogen source for fermentation.

Previously, marine algae were mainly used in the lipids production forbiodiesel. Now, in accordance with the present disclosure, marine algaeis converted to various kinds of bio-based chemicals like succinic acid.There are many advantages to using microalgae, as compared toterrestrial plants, as a feedstock for succinic acid production. Theseinclude no requirement for soil fertility and no need to draw uponvaluable, and often scarce, supplies of freshwater. Moreover, thefixation and storage of CO₂ into the biomass while the microalgae growreduces the concern of greenhouse gas release. Microalgae can be used asthe biomass in lactic acid fermentation after the extraction of lipids.Algae biomass can also be used as a renewable energy feedstock likebio-ethanol or bio-hydrogen.

Microalgae biomass is conventionally formed by four principalbiochemical classes of molecules: carbohydrates, proteins, nucleicacids, and lipids. For carbohydrates, different classes of microalgaeproduce particular types of polysaccharides. Usually, starch consistingmainly of amylose is the energy-storing carbohydrate found in mostalgae. For example, the green alga Tetraselmis suecica accumulates 11%and 47% of its dry weight as starch in nutrient replete and depleteconditions, respectively. Red algae synthesize a carbohydrate polymerknown as floridean starch, consisting mostly of amylose. A commonlyfound polysaccharide in a large number of algal species ischrysolaminarin, a linear polymer of β-1,3 and β-1,6 linked glucoseunits. Different from lignocellulosic biomass, microalgae cells aresingle-cell organisms that are buoyant, evading the need for structuralbiopolymers such as hemicellulose and lignin which, otherwise, areimportant for supporting tissue in land plants.

Besides carbohydrates, microalgae also contain proteins that areimportant enzymes in the regulation of cell metabolism or cross-linkedhydroxyl-proline-rich glycoproteins as the structural cell wall. As withcarbohydrates, lipids serve both as energy reserves and structuralcomponents (membranes) of the cell. The simple fatty acid triglyceridesare important energy reserves. Membranes are mainly constructed fromphospholipids and glycolipids, where the hydrophilic polar phosphate orsugar moieties and the level of saturation of the fatty acyl chainsdetermine the fluidity of the membranes. For some microorganisms,starch-rich cultures may be directly usable as feedstock infermentation, although pretreatment and/or enzymatic digestion could beadvisable to improve the productivity. Additionally, carbohydrate-richmicroalgae residues remaining after lipid extraction can be feedstockfor succinic acid production.

Some strains of microalgae accumulate lipids, such as triglycerides,which can be converted to biodiesel, green diesel, or high-valueoleochemicals. In addition to cost- and energy-efficient methods for therecovery of lipids, a conversion process developed for microalgae shouldbe able to preserve the other major components—carbohydrates andprotein—for profitable uses. Such conversion processes should also beable to directly utilize wet biomass because drying processes areenergy-intense. In certain embodiments, the methods described hereinadvantageously preserve carbohydrates and proteins, and are capable ofutilizing wet biomass. To recover triglycerides and other lipids fromwet material, cell disruption is important. Mechanical disruption iscommonly applied either in the form of intense shear stresses,ultrasonic waves, or electromagnetic fields. Since these forces are mosteffective over short ranges, the slurries are passed through narrow andrestricted zones where intense forces are applied to disrupt the robustmicroalgal cell walls. As a result, processes based on mechanicaldisruption can be energy- and capital-intense as well as difficult toscale up.

Others have previously attempted to use enzymes for digestion of algalcells with the intent to recover lipids and/or carbohydrates. Forexample, cocktails of thermostable enzymes containing up to 14 differentenzymes including several endo- and exo-gluconases have been attempted.While these cocktails produced some lipid recovery, protease activitywas absent in the cocktails and amylase was present only in minorquantities. One cocktail that contained higher amounts of amylase wasrelatively ineffective in facilitating lipid release. Nonetheless, lipidrelease was measured by gravimetric methods which usually overestimatelipid content, and the enzymes used in these attempts were notcommercially available.

Enzymes have been previously used to degrade cell walls, but have notbeen previously shown to enable either recovery of lipids or breakdownof carbohydrates to monomeric sugars. Previous attempts have shown thatamylases did not have any impact on cell wall degradation, resulting ina belief that lysing enzymes from Aspergillus sp. was ineffectivetowards algal cells. In stark contrast to the previous attempts byothers, the present disclosure reveals that protease alone is effectivein digesting algal biomass and releasing lipids that phase-separate orcan be recovered using solvent extraction methods. The lipids can berecovered through solvent extraction with non-polar organic solvents, amixture of polar and non-polar organic solvents, or switchable polaritysolvents, and the lipids can be subsequently converted to fuels oroleochemicals. The protease-alone treatment also partially hydrolyzesalgal polysaccharides to monomeric sugars. Supplementation of theprotease with α-amylase and glucoamylase allows for near-completehydrolysis of carbohydrates to sugars, in addition to simultaneouslyreleasing lipids. The synergistic action of protease and amylasetogether drastically reduces the amount of amylase required forhydrolysis of algal carbohydrates. Furthermore, after removal of lipids,the sugar-rich aqueous phase is easily fermentable by microorganisms,and the residual protein and other cellular material can serve as thenutrient (nitrogen, phosphorus, and other micronutrients) source tosupport microbial growth. As a result, several process options aredescribed, the several processes varying by the biochemical compositionof the algal biomass.

Provided herein are methods of enzymatic hydrolysis that involve asingle-step in which the release of lipids and breakdown ofpolysaccharides into simple fermentable sugars occur simultaneously. Themethods provide for a cost-effective process for the recovery of lipids,sugars, and proteins from microbial biomass by enzymatic digestions.These methods remove the costs associated with drying algae and aseparate extraction of lipids, resulting in a significant cost advantageover methods known in the art. By contrast, in multi-step processes,lipid extraction from dry algae is carried out before the remainingresidue is subjected to enzymatic hydrolysis or acid digestion,producing an economically disadvantageous result. The methods of thepresent disclosure utilize a specific enzyme cocktail comprisingproteases, amylases, or a combination thereof, as opposed to enzymesfrom lignocellulases. This further reduces the costs involved. Incertain embodiments, the depolymerization of all the major algalbiopolymers occurs in a single step through the use of a low-cost andabundant enzyme cocktail containing a mixture of proteases and amylases.

The methods described herein do not involve any acid digestion step.This provides an advantage over other methods which use acid digestionto hydrolyze the carbohydrates. Acid digestion typically producesinhibitors from sugar degradation that inhibit the fermentation process,thereby hindering the conversion of sugars to value-added products. Themethods herein also do not require the presence of free cysteine, asulfur-containing amino acid, in order to activate the enzymes. Theenzymes used herein are active without having to add exogenous cysteineto the digestion medium. Without wishing to be bound by theory, it isbelieved this is due to the action of the particular proteases used. Inone non-limiting example, the proteases digest the microalgae whilesimultaneously hydrolyzing at least 10% of protein and carbohydrates.

An alternative method to release lipids under milder operatingconditions than mechanical methods is through the use of enzymes todigest cells. However, the overall economic viability of enzyme-basedprocesses depends on the cost and ease of enzyme production.Commercially available, low-cost enzymes are thus most desirable. Somecommercial α-amylase exhibits the ability to degrade the glycoproteinswithin cell walls. In addition, alpha-amylase can liquefy theintracellular starch to dextrins, which can be further saccharified tothe sugar monomers by glucoamylase. The enzymatic pretreatment ofmicroalgae biomass for ethanol production by S. cerevisiae S288C needstwo steps of the enzymatic hydrolysis: liquefaction by thermostableα-amylase in 90° C. first at pH 6.0, then saccharification in 55° C. atpH 4.5. The protease from fungi can hydrolyze the glycoprotein, themajor structure in the microalgae cell wall, and there is also someα-amylase and glucoamylase blended in the protease since they are theby-products of the protease in the production by those fungi. Inaccordance with the present disclosure, these mixed enzymes can worktogether to break the cell wall and the carbohydrates at the same time.This process can be performed in low temperatures from 30° C. to 50° C.,and pH from 3 to 5. Moreover, most of the sugar is released in less than1 hour. Since the protease loses the activity quickly (mostly in lessthan 2 hours), it does not bother the microorganism's growth in thefurther fermentation.

Beside the carbon source, nitrogen is an indispensable nutrient for thegrowth and metabolism of microorganism cells. Any kind of nitrogensource feeding (ammonium, free amino acid, urea, or yeast extract) canincrease the fermentation rate and improve the growth of cells. A lackof nitrogen nutrition will lead to the reduction of succinic acidformation rate and yield, and such negative effect cannot be ignoredparticularly when the succinic acid concentration in the medium isrequired at higher levels. In industrial processes, an additionalnitrogen source feeding is necessary and costs a considerable part ofthe total succinic acid cost. Some microalgae biomass contains aconsiderably high level of protein (over 20% in dry weight), which canbe derived to the free amino acid during the pretreatment process byprotease. Therefore, the nitrogen-rich microalgae can serve as thealternative low-cost nitrogen source in the succinic acid fermentation.

In non-limiting examples, Actinobacillus succinogenes is described asthe microorganism in the fermentation, which is flexible in its abilityto efficiently ferment different carbon sources commonly found inhydrolysate, including L-arabinose, cellobiose, fructose, galactose,glucose, lactose, maltose, mannitol, xylose, and others. The metabolicpathways for both hexose and pentose sugars show that these sugars maybe completely converted to mostly succinate, with acetate, formate, andalcohol as the main by-products. It is to be understood, however, thatthe use of microorganisms other than Actinobacillus succinogenes ispossible. Suitable other microorganisms include, but are not limited to:microorganisms of the genus Lactobacillus; microorganisms of the genusPseudomonas; microorganisms of the genus Bacillus; or microorganisms ofthe genus Clostridia, such as Clostridium autoethanogenum, Clostridiumljunclalzlii, or Clostridium ragsdalei.

Unlike the terrestrial lignocellulosic biomass, most microalgae aresingle-celled and do not contain lignin. Some algae species even lackcell walls. This structure allows for much milder conditions and asimpler pretreatment process, which reduces the cost caused by highertemperatures and corrosion in the energy-intensive process. First, themicroalgae cell wall is disrupted by the acid protease, a low costenzyme produced from fungi. In addition, most of their reservedcarbohydrates in the cell are starch-like polysaccharides, which can behydrolyzed by low-cost enzymes such as α-amylase and amyloglucosidase tosugar monomers like glucose, galactose, xylose, or mannose. All of thesereleased sugars can be directly utilized by most of the strain such asthe yeast to produce ethanol, or Actinobacillus succinogenes to producesuccinic acid.

In one aspect, described herein is a method in which an optimizedenzymatic hydrolysis process is used to substitute the conventionalenergy-intensive pretreatment for microalgae biomass. For microalgae,the main obstacle of enzymatic hydrolysis is that intercellular starchgranules are bound within rigid cell walls, requiring a biomasspretreatment step to break down the cell wall and releasepolysaccharides such as starch, structural carbohydrates, and othernutrients, prior to enzymatic hydrolysis and fermentation steps. Thecell wall of microalgae contains glycoproteins as the predominantconstituents in its extracellular matrix. Therefore, degrading thoseproteins is an important step to disrupt the whole cell wall. Someexhibit the protease activity particular to the degradation ofglycoproteins within cell walls. However, using α-amylase only to breakthe cell wall is not quite efficient enough to recover a high sugaryield. To solve this problem, one embodiment of the present disclosurereplaces α-amylase with protease from the same fungi strain (Aspergillusoryzae), which actually also contains a small amount of α-amylase andglucoamylase. This enzyme mixture allows for the full disruption of thecell wall to occur simultaneously to the hydrolysis of thepolysaccharides to monomer sugars. While the microalgae grow to acertain concentration, the microalgae biomass can be collected andconcentrated by settling or centrifugation. In certain embodiments, thepH of the slurry can then be adjusted to about 4.5 by adding citratesalt buffer (50 mM). Then, α-amylase (EC 3.2.1.1) and protease (EC232.752.2) from Aspergillus oryzae and amyloglucosidase (EC 3.2.1.3)from Aspergillus niger are added into the broth to initiate thehydrolysis process. These two enzymes can be added simultaneouslybecause they share a similar range of optimized temperature (30-55° C.)and pH (3.0-5.5).

Using the hydrothermal pretreatment reduces the enzyme loading byincreasing the accessibility of the enzymes to the binding sites onpolysaccharides, which in turn makes the whole process morecost-effective. Similar to the lignocellulosic biomass, applying thehydrothermal pretreatment recrystallizes the cellulose so that thehydrolytic enzymes can easily access the biopolymers. The autoclaveequipment can be applied to the hydrothermal pretreatment up to 120° C.,which is high enough to partially breakdown the cell wall andextracellular matrix of the microalgae. After this treatment, the enzymehas a greater chance to bind the reaction sites on the intracellularpolysaccharides due to the cell wall break. As a result, fewer enzymesare required to reach the same activity. By way of a non-limitingexample, autoclaving at 120° C. for 30 minutes can reduce over 60% ofthe α-amylase loading to achieve the same sugar yield.

In addition, the protein content and other nutrients in the microalgaeare released and soluble after the enzymatic hydrolysis, which can beused as the nitrogen and phosphate source in a fermentation process. Thesolid residue of microalgae biomass after the enzyme hydrolysis can beremoved by centrifugation and used to produce value-added products infurther steps.

Referring now to FIG. 1, provided herein is a method for a two-stageenzymatic hydrolysis of lipid-rich microalgae using proteases andamylases. The block flow diagram in FIG. 1 shows a two-stage processwhere protease is used alone in the first stage for cell disruption andthe recovery of lipids. After treatment with extraction solvents, theaqueous phase is treated with additional amylases, if necessary, tobreak down additional polysaccharides to monomeric sugars. Treatmentwith amylases hydrolyzes carbohydrates into monomeric or oligomericsaccharides. The lipids are recovered from the organic phase throughevaporation. The aqueous phase can be filtered to recover protein-richresidues while leaving behind the soluble sugars, protein, and othercellular material for fermentation into products such as, but notlimited to, alcohols, organic acids, or methane. Alternatively, thewhole slurry can be fermented.

Referring now to FIG. 2, provided herein is a method for a one-stageenzymatic hydrolysis of lipid-rich microalgae using a mixture ofproteases and amylases. The block flow diagram in FIG. 2 shows aone-stage process that incorporates simultaneous treatment with amixture of protease and amylase, and produces sugar-rich hydrolyzate andlipids. In this process, cell digestion and polysaccharide hydrolysisare completed in a single step. Subsequent recovery and conversionpathways are similar to those shown for the two-stage process. Therecovered lipids are capable of use in the production of fuels and otheroleochemicals, while the sugars can be fermented into bio-products suchas, but not limited to, alcohols, organic acids, or methane.

Referring now to FIG. 3, provided herein is an alternate method ofenzymatic hydrolysis of lipid-rich microalgae using either a mixture ofprotease and amylases or protease alone. Following the enzyme treatment,the hydrolyzate is first separated into residual solids and supernatant.The residual solids undergo solvent extraction to recover lipids whilethe supernatant containing hydrolyzed sugars and protein is fermentedinto bio-products such as, but not limited to, alcohols, organic acids,or methane.

Referring now to FIG. 4, provided herein is a method of enzymatichydrolysis of lipid-lean microalgae. These microalgae are rich incarbohydrates but do not contain significant amounts of lipids. For suchmicroalgae, using proteases alone or in combination with amylasesaccomplishes the release of cellular polysaccharides as monomericsugars. Following the enzyme treatment, the hydrolyzate can befermented, with or without prior separation of insoluble solids, intobio-products such as, but not limited to, alcohols, organic acids, ormethane. FIG. 4 shows a block flow diagram for this one-stage process.Thermal treatments can also be used to reduce enzyme loading.

It is to be understood that any suitable solvent extraction can beperformed to achieve separations. A solvent extraction is a method forseparating a substance from one or more other substances by using asolvent, the method relying on variations in the solubilities ofdifferent compounds in the different substances. The substance to beextracted is typically dissolved in a liquid, and a liquid solvent isused for the extraction. A solvent is chosen that does not mix with thecompound in which the substance of interest is dissolved, such that whenleft undistributed, two separate layers will form. Once the solvent isadded, the two liquids may be shaken together for a time and thenallowed to stand for a time until they separate out. The skilledpractitioner will recognize that the choice of solvent will depend onthe chemical and physical properties of all the substances in themixture. In certain methods, the solvent extraction is carried out inseveral stages using different solvents. In one non-limiting example,the solvent is a mixture of hexane and iso-propanol, but many otherpolar or non-polar solvents, or mixtures of polar or non-polar solvents,can be used. Other possible extraction solvents include, but are notlimited to: chloroform, methanol, heptane, hexane, iso-propanol, andmixtures thereof. In some non-limiting examples, a 2:1 (v/v) mixture ofchloroform and methanol is used. In other non-limiting examples, amixture of hexane and iso-propanol is used.

In certain embodiments, the methods described herein are able tosimultaneously accomplish lipid release and carbohydrate breakdown intosimple sugars, in addition to the partial hydrolysis of algal protein.

Though Chlorella species and Schizochitrium limacium are described forexemplary purposes, it is to be understood that the methods describedherein can be performed with any suitable microalgae. There are morethan 50,000 known species of microalgae. Suitable species of microalgaeinclude, but are by no means limited to: species of the Chlorella genus;species of Spirulina genus; Schizochitrium limacium; Botryococcusbraunii; species of the genus Isochrysis, such as Isochrysis galbana;Neochloris oleoabundans; Phaeodactylum tricornutum; Pleurochrysiscarterae; Prymnesium parvum; Scenedesmus dimorphus; Tetraselmis chui;and Tetraselmis suecica. In general, any green algae species can be usedin the methods described herein.

The enzymatic treatment described herein allows for the simultaneousrelease of lipids and breakdown of polysaccharides into simple sugars.In some embodiments, at least 5% of non-polar lipids spontaneouslyseparate from the aqueous phase, allowing for recovery of lipids withoutthe need for an extraction step. In one non-limiting example, at least85% of lipids are extracted and at least 99% of monomer sugars arereleased. In another non-limiting example, at least 20% of protein andcarbohydrates are hydrolyzed. In another non-limiting example, cellularlipids are released while at least 20% of other cellular components aresimultaneously hydrolyzed.

The methods are cost-effective for many reasons, such as being able tobe conducted at low temperatures. The methods are conducted below thetemperature of gelatinization of the substrate. In one non-limitingexample, the temperature is about 50° C. and the pH is about 4.5. In onenon-limiting example of a method with a hydrothermal treatment, anautoclave is used at 120° C. for 30 minutes, and the pretreatmentresults in reducing enzyme loading to ⅓ while still reaching the sameyield of sugar. In one non-limiting example, over 99% of the monomersugars are recovered in less than 2 hours, and over 99% of the lipidsare released.

The aqueous phase from any of the methods can be used as a nitrogensource in a fermentation process to produce products such as, but notlimited to, alcohols, organic acids (like succinic acid), or methane. Insome embodiments, the microalgae residue following enzymatic hydrolysisis used as a nitrogen and phosphate source in succinic acid fermentationwithout an external nutrient. The separated solids recovered from themethods are useful for microbial fermentation, or as animal feed orfertilizer. In some embodiments, lipids become stuck on the solids afterenzymatic digestion. When this occurs, the digested solids can betreated with an organic solvent to recover the lipids.

EXAMPLES Example 1—Recovery of Lipids and Monomeric Sugars from WetAlgal Biomass Using Enzymatic Treatment Methods

Two strains of algae were used in these experiments: a) Chlorella sp.SLA-04, and b) Schizochitrium limacium SR21, obtained from ATCC (MYA1381).

Biomass digestions were performed at a solid concentration of 10% (w/v)in 50 mM citrate buffer adjusted to a pH of 4.5. Protease alone or amixture of protease with α-amylase and amyloglucosidase (all purchasedfrom Sigma) were added to the biomass slurries. Enzyme loadings (perg-biomass) used were as follows—(a) protease 312 U/g; (b) α-amylase 1875U/g; and (c) glucoamylase 18.75 U/g. 1 α-amylase unit is defined as theamount of enzyme that liberates 1 μmol maltose per minute at pH 6.0 and25° C.; 1 glucoamylase unit is defined as the amount of enzyme whichcleaves 1 μmol of maltose per min at pH 4.3 and 25° C.; and 1 proteaseunit is defined as the amount of enzyme which hydrolyzes 1 μmol ofL-leucine-p-nitroanilide per min.

The algae-enzyme mixture was incubated at 50° C. for 6 h in a shakermaintained at 200 rpm. Biomass-free control experiments were alsoperformed. Samples were taken at regular intervals during theexperiments to measure released lipids and sugars.

For lipid recovery and analysis, a 3:2 (v/v) mixture ofhexane/iso-propanol was used. 0.5 mL of the solvent mixture was added to300 μL of the digested samples and extraction was carried out at 90° C.for 30 min. Lipids in the extraction solvent were analyzed andquantified using a gas chromatograph (GC) connected with a flameionization detector (FID). For soluble carbohydrate analysis, thedigested samples were centrifuged and filtered through a 0.22 μmmembrane and the supernatant was analyzed for sugars via HPLC using aShodex SH1011 ion exchange column with refractive index (RI) detector.

In another set of experiments, after enzymatic digestion of SLA-04, thedigested slurry was centrifuged and the residual solids were extractedwith hexane/iso-propanol.

From FIGS. 5A-5B, it can be seen that the rate and extent of lipidreleased from both algae material (SLA-04 and SR-21) is similar intreatments with protease alone as well as in treatments with a mixtureof protease and amylases. For SLA-04, >85% of the lipid (measured asfatty acid methyl ester—FAME) contained in the biomass was released (andextracted) as a result of the enzymatic treatments. For SR-21, >72% ofthe cellular lipid was released. Enzyme-free control treatments did notrelease any extractable lipids.

FIG. 6 shows that at least a portion of the lipids released duringdigestion separated into an oil phase and could be separated without theneed for solvent extraction. FIGS. 7A-7B show that most of the lipidsrecovered after extraction were triglycerides. FIG. 8 shows that atleast a portion of the lipids remained associated with the residualsolids and could be extracted separately. The y-axis in FIG. 8 shows themass of lipids recovered from residual solids relative to the initialmass (feed) of algae biomass.

After enzyme treatment, solubilized monomeric sugar concentrations weremeasured in the digested slurries to determine if carbohydratehydrolysis also occurred during the treatments. FIGS. 9A-9B show thattreatments with protease alone as well as treatments with a mixture ofprotease and amylases resulted in release of monomeric sugars from bothalgal biomass samples (SLA-04 and SR-21). However, the extent of sugarrelease was greater in the treatments that contained amylases. Glucosewas the major sugar recovered along with smaller amounts of xylose. Gelelectrophoresis of the three enzyme preparations (FIG. 10) shows thatthere may be some amylase activity present in the commercial proteasepreparation that may have resulted in some hydrolysis of carbohydrateseven in the absence of the amylases.

Example 2—Sugar Recovery from Microalgal Biomass by Enzymatic Digestion

Enzymatic hydrolysis experiments were conducted using protease,α-amylase, and glucoamylase, and their combinations. A mixed culture oflipid-lean algal biomass (lipid content<5% (w/w)) was obtained from acommercial wastewater treatment facility. The biomass had a carbohydratecontent of 24.5% (w/w). The digestion experiments were performed at asolids' loading of 16% (w/v) at a pH of 4.5. The amount of each enzymewas fixed as follows: 2.5 kU protease, 5 kU of α-amylase, and 150 U ofglucoamylase. Enzymatic hydrolysis reactions were performed in sealedserum vials agitated at 200 rpm for 2 h at 55° C. Samples were collectedat the end of the incubation period and analyzed by HPLC using a ShodexSH1011 ion exchange column with refractive index (RI) detector.

The results (FIG. 11) show that treatments with protease alone resultedin the release of a significant amount of monomeric sugars. Withoutwishing to be bound by theory, it is believed this is possibly due topresence of small amounts of α-amylase and glucoamylase in this enzymepreparation, as previously discussed in Example 1 (FIG. 10).Supplementation of protease with additional α-amylase did not increasesugar yield, indicating that α-amylase activity was not limiting in thissystem. However, addition of glucoamylase to protease or to a mixture ofprotease and α-amylase resulted in near complete breakdown of theavailable polysaccharides to monomeric sugars, indicating thatinsufficient glucoamylase activity was available in both the commercialprotease and α-amylase preparations. Digestion experiments performedwith a mixture of α-amylase and glucoamylase showed better yields thandigestions performed with the individual enzymes. However, lesser yieldswere observed than in treatments that contained protease, indicatingthat either protease activity was necessary for accessing intracellularcarbohydrates or that the α-amylase concentrations used in theseexperiments were insufficient to break down all availablepolysaccharides.

To determine if additional α-amylase (in combination with glucoamylase)results in higher recovery of monomeric sugars, experiments wereperformed at α-amylase loadings between 0.5 and 15 kU. 150 U ofglucoamylase was also added to the solution. Results of theseexperiments (FIG. 13) show that at α-amylase loading below 5 kU, lessthan 60% of the biomass carbohydrates were converted to monomericsugars. However, at an α-amylase loading of 15 kU, nearly 90% of thepolysaccharides were hydrolyzed to sugars. These results show that byappropriately adjusting levels of α-amylase and glucoamylase, nearlycomplete hydrolysis of algal polysaccharides is possible. To determineoptimum pH of this enzyme combination, experiments were performed usinga mixture of 1 kU α-amylase and 150 U glucoamylase at media pH valuesbetween 3 and 6. Results of these experiments (FIG. 13) show that thehighest activity is obtained between pH of 4 and 5.

As discussed above, from the results presented in FIG. 11, algalcarbohydrate hydrolysis can also be accomplished using mixtures ofprotease and glucoamylase. To determine the effect of protease on sugarrecovery, experiments were performed at protease loadings varying from 0to 3 kU and with a fixed glucoamylase loading of 150 U. The results showthat greater than 90% of the algal carbohydrates can be converted intomonomeric sugars at protease loadings of more than 1.25 kU (FIG. 14).

Example 3—Sugar Recovery from Microalgal Biomass by HydrothermalPretreatment Followed by Enzymatic Digestion

Hydrothermal pretreatment was used in combination with enzymaticdigestion to evaluate the potential for lowering enzyme requirements forsugar recovery. For the hydrothermal treatment, algal slurries (16% w/w)were loaded into sealed serum bottles and autoclaved at 120° C. for 30minutes. After cooling to room temperature, the pretreated slurries weredigested at 55° C. for 6 h using a mixture of α-amylase and glucoamylase(pH=4.5). A series of digestions were performed with α-amylase loadingsbetween 0.5 and 15 kU while maintaining a glucoamylase loading of 150 U.Monomeric sugar release was measured at the end of the experiments. Asseen from FIG. 15, the pretreatment improved enzymatic digestibilityrelative to controls which were not thermally pretreated. As a result,up to 90% sugar recovery was able to be achieved at α-amylase loadingsas low as 5 kU.

Example 4—Fermentation of Digested Microalgal Biomass to Succinic Acid

Succinic acid fermentation experiments were conducted for enzymaticallyhydrolyzed algae biomass under 4 conditions with 3 model sugar solutionsas controls. First, 16% (w/v) algal biomass slurries were digested at50° C. for 2 h using a mixture of protease and glucoamylase as describedin Example 2. A portion of the digested slurry was centrifuged (5000rpm, 10 min) to separate out the solids. Fermentations were performedwith the whole digestate as well as with the supernatant both with andwithout yeast extract (2 g/L) as additional nutrient source. Ascontrols, model sugar solutions with the same sugar concentration as thedigestate along with 2 g/L, 5 g/L, or 10 g/L of yeast extract were alsofermented.

Actinobacillus succinogenes type strain 130Z (ATCC 55618) was used asthe fermenting organism. The strain was cultured in tryptic soy brothand a second-generation liquid culture was used as inoculum. MgCO₃ (˜40g/L) was added to the fermentation media as a pH buffer. A 4% (v/v)inoculum was used and fermentations were performed at 37° C. in 50 mLserum bottles that contained sterile carbon dioxide. Soluble nitrogenwas analyzed using a cadmium reduction method, which converted thenitrate to nitrite, as determined by spectrophotometer. Sugars andorganic acids in the samples were analyzed via HPLC.

External supplementation of the hydrolysate with yeast extract showed nosignificant difference on the succinic acid fermentation (FIGS.16A-16B). The succinic acid production from the microalgae hydrolysatewas similar to the model sugar control fermentation containing 10 g/Lyeast extract as the nitrogen source. Thus, the nitrogen andmicronutrients available in the hydrolysate itself were comparable tothose provided by the yeast extract. Hence, for succinic acidfermentation of the algae biomass hydrolysate, no external nitrogensource or other nutrient is required.

TABLE 1 Nitrogen content change in the fermentation After After 24 hoursAfter 24 hours enzymatic fermentation with fermentation withouthydrolysis algae residue algae residue Soluble 27.89 mg/L 21.29 mg/L17.30 mg/L nitrogen content

As shown in Table 1, the soluble nitrogen content remaining after thefermentation of the microalgae hydrolysate with the solid residue washigher than the clarified hydrolysate. This indicates that solublenitrogen is released from the solid residue during the fermentation.

Certain embodiments of the methods and products disclosed herein aredefined in the above examples. It should be understood that theseexamples, while indicating particular embodiments of the invention, aregiven by way of illustration only. From the above discussion and theseexamples, one skilled in the art can ascertain the essentialcharacteristics of this disclosure, and without departing from thespirit and scope thereof, can make various changes and modifications toadapt the methods described herein to various usages and conditions.Various changes may be made and equivalents may be substituted forelements thereof without departing from the essential scope of thedisclosure. In addition, many modifications may be made to adapt aparticular situation or material to the teachings of the disclosurewithout departing from the essential scope thereof.

What is claimed is:
 1. A method of enzymatic hydrolysis for convertingalgal carbohydrates into monomeric sugars, comprising: digestingmicroalgae with a mixture of enzymes in a single step to produce anenzymatically treated biomass, wherein the enzyme mixture comprises atleast one acid protease at an acid protease loading of at least 1.5 kUand at least one glucoamylase; thereafter, separating the enzymaticallytreated biomass into a solid phase and an aqueous phase, wherein theaqueous phase comprises at least monomeric sugars, wherein conversion ofmonomeric sugars from algal carbohydrates is 90% or greater, and whereinthe microalgae is digested at a pH of from 4.5 to 5.5.
 2. The method ofclaim 1, comprising further processing and/or separating one or both ofthe solid phase and the aqueous phase to obtain lipids, proteins, orsolids, or bio-based products.
 3. The method of claim 2, wherein thefurther processing and/or separating comprises subjecting the organicphase to a lipid-solvent separation to recover lipids.
 4. The method ofclaim 2, wherein the further processing comprises: subjecting theaqueous phase to microbial fermentation to obtain a bio-based product.5. The method of claim 4, wherein the bio-based product comprisessuccinic acid.
 6. The method of claim 1, further comprising extractinglipids from the solid phase with an organic solvent.
 7. The method ofclaim 1, wherein the microalgae is lipid-rich wet microalgae.
 8. Themethod of claim 1, wherein at least one of the enzymes is of fungalorigin.
 9. The method of claim 1, wherein the mixture comprises theglucoamylase in an amount of at least 150 U.
 10. The method of claim 1,wherein the mixture further comprises at least one α-amylase.
 11. Themethod of claim 1, wherein the enzymatically treated microalgae has alipid content less than 5% w/w.
 12. A method of obtaining monomericsugars from microalgae, comprising: digesting microalgae with a mixtureof enzymes in one step to produce a digested biomass, wherein the enzymemixture comprises at least one add protease at an aid protease loadingof at least 1.5 kU and at least one glucoamylase; and thereafter,separating the digested biomass into a solid phase and an aqueous phaseusing a filtration step, wherein the solid phase contains lipids and theaqueous phase contains carbohydrates and monomeric sugars; whereinconversion of algal carbohydrates into monomeric sugars is 90% orgreater; and wherein the microalgae is digested at a pH of from 4.5 to5.5.
 13. A method of enzymatic hydrolysis comprising treating microalgaewith enzymes to produce digested biomass, wherein the enzymes comprise amixture of at least one aid protease at an acid protease loading of atleast 1.5 kU and at least one glucoamylase, and separating solids fromliquid in the digested biomass to obtain solids and a supernatant, thesupernatant containing carbohydrates and nutrients, wherein themicroalgae is treated at a pH of from 4.5 to 5.5.